ABSTRACT Genetic crosses coupled with linkage mapping have provided an outstandingly successful approach for locating the genetic determinants of biomedically important traits such as drug resistance and host specificity in P. falciparum malaria. Plasmodium crosses were originally conducted using chimpanzees, but crosses using these primates are now no longer possible. The overall goal of this Program Project grant (P01) is to leverage cutting edge technology that enables us to stage Plasmodium falciparum experimental genetic crosses and isolate large numbers of unique recombinant progeny. We do this using a human-liver chimeric mouse infused with human red blood cells (the FRG huHep/huRBC mouse). We will use this technology to address the emerging health threat posed by the emergence and spread of artemisinin resistant (ART-R) and more recently piperaquine resistant parasites. There has been much fanfare recently about the identification of coding mutations in the kelch13 gene that are strongly associated with ART-R. However, very little is known about the function of this gene and how mutations in kelch13 generate a wide range of resistance levels and fitness effects, and how these effects are compensated by other structural or regulatory changes in the genome. Furthermore, there is evidence of ART- R without mutations in kelch13, and that particular genetic backgrounds are permissive for ART-R. We will use targeted experimental genetic crosses to (i) dissect the genetic complexity of ART-R, (ii) clarify the role of kelch13, (iii) define the regulators and partner genes that control ART-R, and (iv) determine the genetic basis of emerging piperaquine resistance. The project is based in three locations (Notre Dame, Seattle and San Antonio), each with one Research Project supported by a Core facility, with an Administrative Core in Notre Dame. The three research Cores support the tasks of each of the three individual Research Projects, and rely on each other for the generation of progeny lines, sequencing and complementary data analysis. Each Research Project has its own stand- alone research questions, but the flow of information and reagents among projects significantly enhances the potential for discovery that fully leverages the P01 framework. Genetic crosses will be conducted by Core A, while RP01 will increase our understanding of the fundamental aspects of sexual recombination and Mendelian genetics in P. falciparum, allowing us to further optimize methods for generating recombinant progeny. The recombinant progeny will be characterized for drug resistance and competitive growth phenotypes by RP02 and for variation in transcript, protein and metabolite abundance by RP03, with support from Core C. The phenotype, sequence and systems genetic data will be integrated by Core B, which will both conduct analyses and ensure that the archived data will be accessible to all three Research Projects.